PROJECT SUMMARY: Cataracts are the leading cause of blindness in the world and the leading cause of low vision in the United States. Cataracts are devastating to those who have lost their vision, and the only accepted cure, surgery, is a major economic burden to health care. Cataracts occur when large aggregates of cellular material form in the lens and scatter light. Although, there are several pathways in cataract formation, all are associated with aggregation of the structural proteins, called crystallins. We have identified deamidation as a major, if not the major, modification of lens crystallins, during normal aging and cataracts. The most heavily deamidated crystallins are ?B1, ?A3, and ?S. Along with ?A, these proteins are also the major components of the insoluble proteins from aged lenses. In 15 deamidated mimics of the ?-crystallins all, but one, decreases stability and increases the propensity to aggregate. Therefore, we hypothesize that an accumulation of deamidated crystallins is a major cause of cataracts. We propose that deamidation induces aggregation by 1) disrupting structure, 2) altering crystallin-crystallin interactions, and 3) saturating ?-crystallin, the native chaperone in the lens. We have determined that deamidation, at critical interfaces within ?-crystallin dimers, creates solvent accessible pockets that decrease stability and increase susceptibility to proteolysis and UV-B damage. Deamidation, on surfaces, alters interactions between ?-crystallins. And, deamidation causes aggregation more readily during crystallin unfolding preventing rescue by ?-crystallin. In this proposal, we will determine the protective protein interactions in the lens that prevent aggregation of deamidated crystallins. We will determine the functional significance to our recent finding that ?-crystallins are not static, but have different conformations in hetero-oligomers. We will determine the role of a flexible loop we have recently identified to be a potential site of crystallin interactions. And, we will solve structures of ?- and ?- complexes. We will use state-of-the-art NMR, hydrogen deuterium exchange with high-resolution mass spectrometry, and cryo-electron microscopy using high-end instruments not found elsewhere. We will screen for the most disruptive deamidation sites using expressed deamidated mimics with lens extracts from human donor lenses and by over-expressing mimics in zebrafish, including in a ?A-crystallin knock-out zebrafish. We will directly link deamidation with aggregation in vivo. The goal is to understand complex interactions between crystallins in order to stabilize them and rescue deamidated crystallins from aggregation. Deamidation has also been detected in other protein aggregation diseases, such as Alzheimer's, and our findings will provide insight into a potential shared mechanism in these more devastating neurological diseases.